Oesophageal squamous cell carcinoma (OSCC) is a major health burden in Sub-Saharan Africa, and novel chemotherapies are urgently required to combat this disease. The heat shock protein 90 (HSP90) inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) has previously been proposed as a possible candidate drug.
Trang 1R E S E A R C H A R T I C L E Open Access
Use of NQO1 status as a selective biomarker for oesophageal squamous cell carcinomas with
greater sensitivity to 17-AAG
Katie E Hadley and Denver T Hendricks*
Abstract
Background: Oesophageal squamous cell carcinoma (OSCC) is a major health burden in Sub-Saharan Africa, and novel chemotherapies are urgently required to combat this disease The heat shock protein 90 (HSP90) inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) has previously been proposed as a possible candidate drug NADPH quinone oxidoreductase 1 (NQO1) is known to increase the potency of 17-AAG, therefore we investigated the effects of 17-AAG in OSCC cell lines in the context of their NQO1 status
Methods: We used MTT assays to compare the sensitivity of a panel of OSCC cell lines to 17-AAG Western blotting, and RT-PCR were used to investigate NQO1 protein and mRNA levels, while an RFLP approach was used to investigate the NQO1 C609T SNP
Results: Expression of NQO1 markedly increased sensitivity to 17-AAG in the OSCC cell lines, while normal fibroblasts, which expressed HSP90 at much lower levels, were more resistant to 17-AAG In isolation, neither the C609T SNP, nor NQO1 mRNA levels was an accurate predictor of NQO1 protein levels
Conclusions: Since NQO1 greatly enhances the anti-cancer effects of 17-AAG, this could be used as a selective marker for patients that would benefit most from 17-AAG chemotherapy at low doses Testing for the presence of the C609T SNP in both alleles could be used as a screen to exclude potentially poor responders to 17-AAG treatment at low dosages
Background
OSCC presents a major health burden in Sub-Saharan
Africa, and novel chemotherapies are urgently needed to
combat this disease HSP90 has been shown to be
overex-pressed in a number of cancers, and presents an attractive
target for anti-cancer therapy, as it plays a central role in
contributing to the maintenance of a number of the
charac-teristic hallmarks of cancer cells, by chaperoning key
pro-teins, and maintaining active conformations of signalling
proteins, reviewed in [1] These include important
signal-ling proteins like EGFR and IGF1-R that have been
impli-cated in sustaining the neoplastic phenotype in OSCC [2]
Different HSP90 inhibitors have been shown to have
promise as chemotherapeutics
These include the family of benzoquinone ansamycins
(BA’s), such as geldanamycin and its derivatives 17-AAG
and 17-DMAG It has been suggested that 17-AAG could
be useful for treatment of OSCC [2] There are several drawback to this class of drugs, which are reviewed in [3], most notably the induction of hepatotoxicity This results from one electron reduction by members of the cyto-chrome p450 family This reaction results in unstable inter-mediates, damaging the tissue, which manifests as severe hepatotoxicity [4] On the other hand, the benzoquinone ansamycins can also undergo two electron reduction by the enzyme NADPH quinone oxidoreductase 1 (NQO1), which results in a compound with higher affinity for HSP90, which is therefore a more potent inhibitor [5,6] The gene encoding NQO1 has been found to contain
a single nucleotide polymorphism at position 609 The C609T SNP causes a proline to serine mutation at position
187 [7], allowing ubiquitination of NQO1 and reducing sta-bility of the protein [8] Thus, the SNP is effectively a null mutation, as patients homozygous for T at position 609 will express NQO1 that is rapidly degraded by the proteasome
* Correspondence: denver.hendricks@uct.ac.za
Division of Medical Biochemistry, Department of Clinical Laboratory Sciences,
Faculty of Health Sciences, University of Cape Town, Anzio Road,
Observatory, Cape Town 7925, South Africa
© 2014 Hadley and Hendricks; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this
Trang 2We aimed to examine the potential of 17-AAG as a
chemotherapeutic drug for OSCC, using a panel of cell
lines with different NQO1 levels Results indicated that
NQO1 status could be an important determining factor in
tumour response to 17-AAG We next investigated whether
the presence of NQO1 enzyme could be predicted with
either the absence of the C609T SNP, or expression
levels of NQO1 mRNA Although neither factor alone
was sufficient, SNP analysis could allow exclusion of a
cohort of NQO1-negative patients who would be less
sensitive to 17-AAG
Methods
Cell lines, plasmids, transfections and drugs
17-AAG was purchased from Calbiochem The human
OSCC cell lines WHCO1 and WHCO6, derived from
South African patients, were a gift from Prof R Veale, and
described in [9] The Kyse cell lines were purchased from
DSMZ, Germany All cells were grown in DMEM with
10% FCS, in the presence of penicillin and streptomycin
The plasmids for overexpression of NQO1
(pEFIRES-empty, and pEFIRES-NQO1) were a kind gift from Yosef
Shaul (Weizmann Institute of Science) [10] Cells were
transfected using Transfectin (BioRad) and transfected cells
were selected using puromycin (Calbiochem) Pools
of stably transfected cells were maintained in 1.5 μg/ml
puromycin
MTT assay
Cells were plated in 96 well plates at a density of 5000
cells per well The following day, cells were treated with
drug at different concentrations After 2 or more days of
incubation, 10μl of sterile MTT solution (5 mg/ml in PBS)
was added to each well, and plates were incubated for
4 hours Thereafter, 100μl of solubilisation reagent (10%
SDS, 0.01M HCl) was added to each well Plates were
in-cubated at 37°C overnight, before the absorbance was
measured at 595 nm
Western blotting
Proteins were harvested in RIPA buffer, and sonicated
for 10s Protein concentration was calculated using the
BCA kit (Pierce) Equal amounts of protein were separated
on a polyacrylamide gel, and transferred to a nitrocellulose
membrane (Amersham) Membranes were blocked in 5%
fat free milk powder, before incubation with the
follow-ing primary antibodies: NQO1 A180 (sc32793); GAPDH
0411 (sc47424);β tubulin H235 (sc9104); PARP 1/2 H250
(sc7150) (all from Santa Cruz Biotechnology)
SNP analysis
Genomic DNA was harvested from cell lines using
Qiazol, according to the user defined protocol provided on
the manufacturer’s website PCR was performed using
Amplitaq Gold (Applied Biosystems), and primer sequences from [11] PCR products were purified using Wizard SV Spin columns (Promega) before being digested overnight with Hinf1 (Thermo Scientific) Digested DNA fragments were analysed by polyacrylamide gel electrophoresis, stain-ing with ethidium bromide
Quantitative RT-PCR Total RNA was harvested from cells at approximately 60- 80% confluency using the Qiazol reagent (Qiagen), according to the manufacturer’s instructions After agar-ose gel electrophoresis to confirm RNA integrity, 1μg was reverse transcribed using random hexamer primers, and Impromtu RTase (Invitrogen) cDNA was submitted to quantitative RT-PCR using Sybr-fast reaction mix (Kapa Biosystems), and primers for HSP90α (F: TGAGGACAG ACACAGGTGAAC 3′ and R: TGGTCCAGATGGGCTT TGTT 3′) NQO1 (F: TGAAGAAGAAAGGATGGGAG G3′ and R: AGGGGGAACTGGAATATCAC 3′) and β actin (F: AGGAAGGAAGGCTGGAAGAG 3′ and R: AT CGTGCGTGACATTAAGGAG3′) β actin was used as
a housekeeping gene Relative expression was calculated using comparative Ct values Results of two to three inde-pendent experiments were pooled
Statistical analysis GraphPad Prism software was used for statistical analysis,
as indicated in figure legends For MTT dose response assays, absorbance values were analysed by nonlinear re-gression, with a sigmoidal curve (variable slope), allowing calculation of the IC50 value Dose response experiments were repeated several times in each cell line, and data were pooled to give a more accurate estimation of the IC50and 95% confidence intervals around the value
Results
NQO1 enhances sensitivity of OSCC cell lines to 17-AAG
We analysed the response of a panel of OSCC cell lines
to 17-AAG Using dose response MTT assays, we estab-lished the IC50 concentrations of 17-AAG for each cell line We noticed that all the cell lines in the panel were relatively sensitive to 17-AAG, with IC50 values in the sub-micromolar range (Figure 1) However, five of the OSCC cell lines were significantly more sensitive, with
IC50 values below 120 nM On further investigation, we found that the sensitivity to 17-AAG correlated very well with endogenous expression of NQO1, as detected by Western blotting (Figure 1A, bottom panel) Cell lines with detectable levels of endogenous NQO1 were mark-edly more sensitive to 17-AAG
In order to confirm that the levels of NQO1 were indeed responsible for the differences in sensitivity to 17-AAG, we generated stable cell lines overexpressing NQO1 (WHCO1 pEF-NQO1) or the empty vector (WHCO1 pEF-empty)
Hadley and Hendricks BMC Cancer 2014, 14:334 Page 2 of 8 http://www.biomedcentral.com/1471-2407/14/334
Trang 3Overexpression of NQO1 was confirmed by Western
blot-ting, and NQO1 levels were found to be similar to the levels
of endogenous NQO1 in the cell lines in which NQO1 was
detectable (Additional file 1: Figure S1) Analysis of the
sen-sitivity of the stable cell lines to 17-AAG revealed that
over-expression of NQO1 resulted in increased sensitivity to the
drug, reducing the IC50 value of the cells from 0.618μM
(WHCO1 pEF-empty) to 0.0738μM, which is in line with
the IC values of cell lines that express detectable NQO1
(Figure 1A) We further confirmed the increased sensitivity
of the cells by investigating PARP cleavage, a marker of apoptosis, in response to 17-AAG While WHCO1 cells transfected with empty vector only exhibited PARP cleavage after treatment with 1 μM 17-AAG for 24 hours, NQO1 transfected cells exhibited PARP cleavage at the lower con-centration of 0.1μM 17-AAG (Figure 1C )
We noted that NQO1 protein levels decreased in the presence of increasing concentrations of 17-AAG A
Figure 1 NQO1 increases sensitivity of OSCC cell lines to 17-AAG (A) OSCC cell lines were analysed by dose response MTT assay, and IC 50
concentrations were established for each cell line, by pooling 2 to 6 independent experiments The IC 50 value is presented in the histogram, with error bars representing the 95% confidence interval around this value The lower panel shows a Western blot of NQO1 levels, with β tubulin as a loading control The Western blot is representative of at least two independent experiments (B) WHCO1 cells were stably transfected with pEF-empty
or pEF-NQO1 Dose response curves were performed in triplicate (error bars represent SEM) Results are representative of two independent experiments (C) Cells were treated with different concentrations of 17-AAG for 24 hr, and protein was harvested and analysed for PARP cleavage by Western blot NQO1 overexpression was also confirmed by Western blot Results are representative of two independent experiments.
Trang 4similar effect was observed with endogenous NQO1 in
Kyse 70 and Kyse 150 cells (Additional file 2: Figure S2)
However, we did not detect a significant downregulation
of NQO1 mRNA brought about by treatment with
17-AAG (Additional file 2: Figure S2), suggesting that the
observed downregulation at the protein level is a
post-transcriptional event
We selected cell lines with either detectable or
undetect-able levels of endogenous NQO1, and examined their
pro-liferation over several days in the presence of increasing
concentrations of 17-AAG (Figure 2) Although none of
the cell lines showed proliferation in the presence of 1μM
17-AAG, we observed a distinct dichotomy between those
OSCC cell lines which expressed NQO1 (Kyse 70 and
Kyse 150), which did not proliferate in the presence of
0.1μM 17-AAG, and those in which NQO1 was not
de-tectable (WHCO1 and Kyse 30), which displayed
prolif-eration levels similar to untreated cells in the presence
of 0.1μM 17-AAG Western blotting for PARP cleavage
as a marker of apoptosis showed that at 0.1μM 17-AAG,
apoptosis was induced within 24 hr of treatment in Kyse
150, and 72 hr of treatment in Kyse 70 (Additional file 3: Figure S3A) No induction of PARP cleavage was de-tectable in WHCO1 or Kyse 30 at this concentration of 17-AAG over a similar time frame (Additional file 3: Figure S3 B)
Interestingly, the normal fibroblasts DMB and FG0, were relatively unaffected by the presence of 0.1μM 17-AAG, and proliferated at a similar rate to untreated cells (Figure 2) This is despite their having detectable levels
of the 17-AAG metabolising enzyme NQO1, similar to the levels observed in Kyse 70 and Kyse 150 (Figure 3A) This highlights the selectivity of 17-AAG for cancer cells, presumably due to the increased reliance of cancer cells on HSP90 [12] As expected, we observed that the expression
of HSP90 (α subunit) is significantly higher in the OSCC cell lines tested than the normal fibroblasts (Figure 3B), indicative of their increased reliance on HSP90 as a chaperone This suggests that in NQO1 expressing pa-tients, treatment with a low dose of 17-AAG could still selectively target cancer cells and have minimal effects
on normal cells, even though they may express NQO1
WHCO1
0.0 0.5 1.0 1.5
2.0
UT 0.1 M
1 M
Day
Kyse 30
0.0 0.5 1.0 1.5
2.0
UT 0.1 M
1 M
Day
Kyse 70
0.0 0.2 0.4 0.6
0.8
UT 0.1 M
1 M
Day
Kyse 150
0.0 0.5 1.0 1.5
2.0
UT 0.1 M
1 M
Day
DMB
0.0 0.1 0.2 0.3 0.4
0.5
UT 0.1 M
1 M
Day
FG 0
0.0 0.2 0.4
0.6
UT 0.1 M
1 M
Day
Figure 2 Sensitivity of OSCC cell lines and normal fibroblasts to 17-AAG OSCC cell lines were plated in 96 well plates, and their proliferation was measured over several days in the presence of increasing concentrations of 17-AAG using a MTT assay Each point was performed in triplicate, and error bars represent standard deviation Results are representative of two independent experiments.
Hadley and Hendricks BMC Cancer 2014, 14:334 Page 4 of 8 http://www.biomedcentral.com/1471-2407/14/334
Trang 5NQO1 protein levels in OSCC cell lines depend on C609T
SNP and expression levels of NQO1 mRNA
Since the presence of NQO1 was an indicator of high
sensitivity to 17-AAG, we postulated that this could be a
useful marker of a patient’s suitability for treatment with
low doses of 17-AAG We sought to investigate whether
the presence or absence of the NQO1 C609T SNP could
allow rapid identification of cell lines with high NQO1
levels, in the hope that this may ultimately be extended
to a clinical setting, for selection of patients who would
likely respond better to 17-AAG We used an RFLP
ap-proach to genotype the panel of cell lines used (Figure 4)
We found that all of the cell lines in which NQO1 was
detectable had at least one WT allele (CC or CT) Two
cell lines homozygous for the C609T SNP (Kyse 30 and
Kyse 450) did not express detectable NQO1, which is
consistent with this SNP allowing increased turnover
of the nascent protein Unexpectedly, we observed that
two cell lines (WHCO1 and WHCO6) with
undetect-able NQO1 levels, were homozygous for the wild-type
(C) allele Thus in these cell lines, the absence of
detect-able NQO1 could not be accounted for by more rapid
protein degradation caused by the C609T SNP
In an attempt to explain this unexpected result, we
ex-amined NQO1 mRNA expression in the panel of OSCC
cell lines using real time PCR We found that WHCO1
and WHCO6 expressed lower levels of NQO1 mRNA
than the other cell lines with at least one C allele
(approxi-mately 50-100 fold lower than Kyse 70, 150, 180 and 520)
The expression of NQO1 in WHCO1 and WHCO6 was
also approximately 25 times lower than in the two cell
lines identified as homozygous for the C609T SNP The
lower levels of NQO1 mRNA could possibly account for
the undetectable levels of endogenous NQO1 protein in
WHCO1 and WHCO6
Figure 3 NQO1 levels and HSP90AA1 expression in OSCC cell lines and normal fibroblasts (A) Protein lysates of OSCC cell lines and normal fibroblasts (DMB and FG 0 ) were analysed by Western blot for endogenous levels of NQO1 Results are representative of 2 independent experiments (B) Real time PCR was used to measure levels of HSP90AA1 mRNA relative to the housekeeping gene β actin, in a panel of OSCC cell lines and normal fibroblasts Histogram shows pooled results of 2- 3 independent experiments, analysed by one-way ANOVA, with Dunnet ’s post-test relative to WHCO1.
Figure 4 NQO1 C609T SNP and NQO1 mRNA levels contribute
to endogenous levels of NQO1 protein Genomic DNA from a panel of OSCC cell lines was amplified using primers spanning the C609T SNP PCR products were digested with HINf1 α, and analysed
by polyacrylamide gel electrophoresis to establish the presence of the SNP (B) Total RNA was extracted from cells at approximately 80% confluency RNA was reverse transcribed and the abundance
of NQO1 mRNA was analysed relative to β actin mRNA using real time PCR Histogram shows pooled results of 2- 3 independent experiments, analysed by one-way ANOVA, with Dunnet ’s post-test relative to WHCO1.
Trang 6Our results show a clear correlation between NQO1
levels and sensitivity to 17-AAG as expected [5] Although
NQO1 activation is considered necessary for 17-AAG
activity [3], even OSCC cell lines without detectable
NQO1 showed considerable sensitivity to 17-AAG, with
IC50 concentrations around 1μM Although this might
lead one to overestimate the promise of 17-AAG as a
chemotherapeutic option for OSCC, one must be
mind-ful of the limitations of this in vitro study Firstly, we
have not measured the negative side effects attributed to
the drug Indeed, a recent clinical trial of 17-AAG found
that the severity of the side effects outweighed the clinical
benefit to patients with solid tumours [13] However, this
clinical trial did not investigate NQO1 levels in the
en-rolled patients, nor has any trial testing 17-AAG done so,
to the best of our knowledge This may have important
implications as we describe below Secondly, there are
indications in the literature that cultured cell lines may
express higher levels of NQO1 than lung and colon
pa-tient tumour tissue [14] We have not been able to directly
compare NQO1 levels in cell lines and OSCC tumour
tissue, but in vivo expression may well not be as high as
that observed in the cultured cell lines
The findings of this report suggest that if patients could
be stratified on the basis of NQO1 protein levels, then
OSCC patients expressing NQO1 could potentially benefit
from administration of low doses of 17-AAG, possibly
in combination with other chemotherapeutics This is
because NQO1 positive patients would likely be
respon-sive to much lower concentrations of the drug The low
dose of 17-AAG would limit the extent of toxic side
ef-fects experienced, as observed in clinical trials, where at
the six lowest doses administered (6- 125 mg/m2), only
one out of 20 patients experienced dose-limiting toxicity,
compared with eight out of fifteen patients on the two
highest doses (175- 225 mg/m2) [13] Since severe
hepato-toxicity resulting from 17-AAG treatment is reported to
be due to metabolism by a different family of reductases
[4], this is unlikely to correlate with NQO1 expression,
although this would need to be confirmed in vivo
Further-more, very low concentrations of 17-AAG would likely
have minimal effect on normal cells, even those expressing
NQO1, due to their much lower reliance on HSP90
How-ever, there is a clear need for further in vivo testing to
con-firm that the presence or absence of NQO1 does not affect
hepatotoxicity, and that severe side-effects can be mitigated
by administration of sufficiently low doses
An alternative possibility may be the approach proposed
by Karkoulis and co-workers [15] for the treatment of
bladder cancer These authors propose that the negative
side effects of BA chemotherapeutics (in this case
gelda-namycin) may be mitigated by orthotopic
administra-tion of drug In the case of OSCC, similar to bladder
cancer, the tumour site is relatively accessible; therefore
an orthotopic delivery may also be feasible This would allow exposure to dosages that effectively target the tumour, without reaching systemic concentrations that cause hepatotoxicity
We noticed that 17-AAG treatment resulted in a dose-dependent decrease in endogenously and exogenously expressed NQO1 This is similar to the effect reported by Gaspar and co-workers [16] who suggested that this down-regulation of NQO1 by 17-AAG may play a role in acquisi-tion of resistance to the drug We found that there was no down-regulation of NQO1 mRNA levels (Additional file 2: Figure S2), suggesting a post-transcriptional mechanism of control It is not clear what this mechanism may entail, since disruption of HSP90 activity affects a wide range of cellular functions Although there is no evidence support-ing a direct interaction between NQO1 and HSP90, NQO1 levels are reported to depend heavily on FAD levels [17], which may be disrupted by HSP90 inhibition
It was interesting to note that the absence of detectable NQO1 in two of the cell lines (WHCO1 and WHCO6) could not be accounted for by the presence of the C609T SNP, but rather seemed to correlate with low expression
of the NQO1 gene Further investigation in tumour sam-ples could shed light on whether this accurately reflects NQO1 expression in patients, or whether it is an artefact
of a subset of cultured cell lines The possibility therefore exists that expression of NQO1 could be induced in these two cell lines under particular environmental circum-stances, such as those which may be experienced in cells
of a solid tumour, e.g the presence of reactive oxygen spe-cies or hypoxia We postulate that due to the possibility of induction of the gene in a tumour setting, it will be neces-sary to specifically investigate NQO1 protein levels in biopsies, in order to estimate potential sensitivity to 17-AAG This could be done using protein detection (Western blot/immunohistochemistry), or using an NQO1 enzyme activity assay However, the SNP could be used as a rapid test to exclude patients with a TT genotype, who would not express NQO1 and would therefore be poor candidates for 17-AAG treatment
The relevance of NQO1 levels in the clinical setting has been discussed by Siegel et al [18] The authors make the point that NQO1 levels and activity may not remain stable over the course of the treatment, limiting the predictive value of a protein assay, and supporting use of the SNP as
a better biomarker of 17-AAG responsiveness If the SNP were used as a biomarker for responsiveness, patients with the homozygous null mutation, who will certainly not express active NQO1 could easily be excluded from 17-AAG treatment While SNP analysis could provide a relatively simple tool for elimination of non-expressors, some patients with the wild-type genotype may also ex-press low levels of the protein, and also be less sensitive
Hadley and Hendricks BMC Cancer 2014, 14:334 Page 6 of 8 http://www.biomedcentral.com/1471-2407/14/334
Trang 7to 17-AAG treatment Thus we propose that 17AAG may
still hold promise as a chemotherapy, under certain
condi-tions These include that the drug either be administered
orthotopically, or at low concentrations, using the C609T
SNP as a screen to exclude non-expressors of NQO1 who
would be poor responders
Conclusions
Despite the known side effects of 17-AAG, the extreme
sensitivity of NQO1-expressing cell lines to 17-AAG,
com-pared to normal cells or NQO1 negative cells, suggests that
this drug could be a useful chemotherapeutic for NQO1
positive OSCC tumours, due to the much lower
concentra-tion required for anti-cancer activity The presence of the
C609T SNP in both alleles could be used as a screen to
exclude potentially poor responders to 17-AAG treatment
at low dosages This warrants further investigation in an
in vivo model
Additional files
Additional file 1: Figure S1 Expression of NQO1 in stably transfected
cells is similar to that of endogenous NQO1 in cell lines in which this is
detectable Whole cell lysates were analysed by western blotting for
NQO1, with β-tubulin as a loading control.
Additional file 2: Figure S2 17-AAG causes a decrease in endogenous
NQO1 levels Kyse 70 and 150 were treated for 24 h with increasing
concentrations of 17-AAG (A) Cellular protein was harvested and NQO1
levels were determined by Western blotting β tubulin serves as a loading
control (B) Real time PCR was used to measure levels of NQO1 mRNA
relative to the housekeeping gene β actin Histogram shows pooled
results of 2- 4 independent experiments, analysed by one-way ANOVA,
but found to be non-significant.
Additional file 3: Figure S3 17-AAG causes cell death by apoptosis.
(A) Kyse 70 and Kyse 150 cells were treated with 100 nM 17-AAG for different
time periods Total protein lysates were analysed by Western blotting for
the presence of cleaved PARP, a marker of apoptosis β tubulin was used
as a loading control (B) WHCO1 and Kyse 30 were treated with 100 nM
17-AAG for 96 h and whole cell lysates were analysed as described above.
Abbreviations
17-AAG: 17-N-allylamino-17-demethoxygeldanamycin; BA: Benzoquinone
ansamycin; EGFR: Epidermal growth factor receptor; HSP90: Heat shock
protein 90; FAD: Flavin adenine dinucleotide; IGF-1R: Insulin-like growth
factor 1 receptor; MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide; NQO1: NAP (P) H: quinone oxidoreductase 1; OSCC: Oesophageal
squamous cell carcinoma; PARP: Poly ADP ribose polymerase; RT-PCR: Real
time polymerase chain reaction; RFLP: Restriction fragment length
polymorphism; SNP: Single nucleotide polymorphism.
Competing interests
The authors declare that they have no competing interests.
Authors ’ contributions
KEH performed all the experiments and drafted the manuscript DTH
participated in experimental design and helped draft the manuscript Both
authors read and approved the final manuscript.
Acknowledgements
The authors would like to thank all members of the Cancer lab, especially
Prof Virna Leaner for helpful discussions and suggestions We also thank
Catherine Whibley, Laja Osoniyi and Allison Carter for their initial
contribution to this project We are also grateful to Prof Shaul for kindly
donating expression plasmids This work was supported by funding from CANSA, MRC and UCT and a postdoctoral fellowship to KH from NECSA Received: 4 October 2013 Accepted: 23 April 2014
Published: 15 May 2014
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doi:10.1186/1471-2407-14-334
Cite this article as: Hadley and Hendricks: Use of NQO1 status as a
selective biomarker for oesophageal squamous cell carcinomas with
greater sensitivity to 17-AAG BMC Cancer 2014 14:334.
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